Eliciting neutralizing Abs against the native (fusogenic) trimer of the HIV-1 envelope glycoproteins (Env) is problematic due to lability in the constituent subunits, gp120 and gp41, which leads to the elicitation of mainly non-neutralizing antibodies against irrelevant forms of Env. Therefore, a means of engineering and producing a stable and homogeneous preparation of native Env trimers is likely to be beneficial for HIV-1 vaccine design. Rational approaches have been met with limited success, due in part to a lack of information about the structural details of the trimer. Our approach does not strictly require detailed knowledge of Env trimer structure. In the R21 phase, in vitro mutagenesis will be employed to introduce sequence diversity into targeted regions within env, and the env `libraries' subcloned en masse into a HIV-1 expression vector for the production of `HIV-1 display Env libraries' (i.e. HIV-1 populations whose diverse members are each distinguished by a unique env gene sequence and the cognate copies of Env it displays on its surface). The HIV-1 display Env libraries will then be selected on the basis of resistance to heat, chemical denaturants and CD4-receptor destabilization, and the surviving virions rescued by infecting target cells. Multiple rounds of selection may be expected to yield HIV-1 that display native Env trimers of enhanced stability. We will also try depleting the HIV-1 Env libraries of clones that are particularly reactive with non-neutralizing monoclonal antibodies while enriching for those clones with diminished display of immunodominant and irrelevant epitopes. The in vitro-selected, hyperstable HIV-1 Env variants will be used as immunogens to determine their potential as HIV-1 vaccine leads. R33 phase immunization studies will be accompanied by high precision serum Ab specificity mapping to guide Env immunogen optimization. Finally, a specifically tailored screen for soluble and natively folded gp140s will be performed that uses the stability-selected HIV-1 Env variants as input. PUBLIC HEALTH RELEVANCE: We aim to select mutants of HIV in which key molecules on the viral surface are more stable than that found in nature. Inactivated forms of such mutant viruses may elicit more effective immune (antibody) responses and may therefore lead to better vaccines against HIV/AIDS.